Human histidine triad nucleotide binding protein 1 (hHint1) has emerged as a target of interest due to its involvement in the regulation of a broad range of CNS functions, including opiate signaling, tolerance, neuropathic pain and nicotine dependence.1,2 Human hHint1 belongs to the histidine triad (HIT) superfamily which are characterized by their conserved sequence motif, His-X-His-X-His-XX, where X is a hydrophobic residue. Human hHint1 exists as a homodimer and possesses nucleoside phosphoramidase and acyl-AMP hydrolase activity, with a substrate preference for purine over pyrimidine nucleosides.3 Structural and kinetic studies have shown that hHint1 possess two identical and independent nucleotide-binding subunits.4-6 Each monomer consists of two alpha helices. A conserved string of hydrophobic residues in or adjacent to the β-sheets creates a binding pocket (S1) for the nucleobase, while the aspartate (43) residue anchors the ribose sugar. The α-monophosphate group interacts with a conserved string of polar residues including the partially positive His114 and the nucleophilic His112 in the active site.
The side chains of the nucleoside phosphoramidates or acyl-AMP can occupy a relative shallow and solvent accessible pocket containing the only tryptophan residue in hHint1. A nucleophilic histidine (His112) residue forming part of the active site triad of hHint1 is responsible for the catalysis. A detailed investigation of the kinetic mechanism of hHint1 has revealed that the mechanism proceeds by rapid formation of the nucleotidylated-His intermediate, followed by partially rate limiting, water mediated hydrolysis and subsequent release of the nucleoside monophosphate from the active site.5 The nucleoside phosphoramidase activity of hHint1 has been shown to be necessary for the activation of several preclinical and clinically approved antiviral and anticancer phsophramidate pronucleotides.7-10 In addition, Chou and Wagner et al. have demonstrated that lysyl t-RNA synthetase generated lysyl-AMP is also a substrate for hHint1 in vitro.11 
Hint proteins are highly conserved across all the kingdoms of life, suggesting that they have an important biological function. Hint1 has been implicated in the regulation of MITF/USF2 transcriptional activation complex in mast cells,12 t-RNA synthetase amino acid adenylation,11 apoptosis13 and tumorigenicity.14 
Hint1 is widely expressed in the region of brain primarily responsible for the modulation of pain [periaquaductal grey area (PAG)], addiction properties (nucleus accumbens) and the motor and sensory functions (cerebral cortex).1 In agreement with these results, alterations in gene function or aberrant expression of hHint1 have been found in the brain tissues of clinical patients suffering from schizophrenia and bipolar disorders.15 Moreover, Hint1−/− mice have been shown to exhibit hypersensitivity to amphetamine and decreased dependence on nicotine in self-administration studies.16,17 
In addition, the NMDAR mediated feedback inhibition of the analgesic response has been demonstrated to be critically dependent on the co-association of Hint1 with MOR and NMDAR.18 Hence, Hint1−/− mice have shown to display an enhanced analgesic response Δδ=1.11 ppm indicating that nucleobase recognition maybe a key event in driving the molecular recognition of nucleotide based ligands by Hint1.
Given its involvement in, among other things, apoptosis, tumorigenicity, and pain modulation, new agents are desirable for the modulation (e.g., inhibition) of hHint1.